T cell receptor-deficient t cell compositions

ABSTRACT

The invention is directed to modified T cells, methods of making and using isolated, modified T cells, and methods of using these isolated, modified T cells to address diseases and disorders. In one embodiment, this invention broadly relates to TCR-deficient T cells, isolated populations thereof, and compositions comprising the same. In another embodiment of the invention, these TCR-deficient T cells are designed to express a functional non-TCR receptor. The invention also pertains to methods of making said TCR-deficient T cells, and methods of reducing or ameliorating, or preventing or treating, diseases and disorders using said TCR-deficient T cells, populations thereof, or compositions comprising the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. provisionalpatent application No. 61/255,980, filed Oct. 29, 2009, the disclosureof which is herein incorporated by reference in its entirety.

This invention was made with government support under contract number CA130911 awarded by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention

The invention is directed to TCR-deficient T cells, methods of makingand using TCR-deficient T cells, and methods of using theseTCR-deficient T cells to address diseases and disorders. In oneembodiment, the invention broadly relates to TCR-deficient T cells,isolated populations thereof, and compositions comprising the same. Inanother embodiment of the invention, said TCR-deficient T cells arefurther designed to express a functional non-TCR receptor. The inventionalso pertains to methods of making said TCR-deficient T cells, andmethods of reducing or ameliorating, or preventing or treating, diseasesand disorders using said TCR-deficient T cells, populations thereof, orcompositions comprising the same.

Description of Related Art

The global burden of cancer doubled between 1975 and 2000, and cancer isexpected to become the leading cause of death worldwide by 2010.According to the American Cancer Society, it is projected to doubleagain by 2020 and to triple by 2030. Thus, there is a need for moreeffective therapies to treat various forms of cancer. Ideally, anycancer therapy should be effective (at killing cancerous cells),targeted (i.e. selective, to avoid killing healthy cells), permanent (toavoid relapse and metastasis), and affordable. Today's standards of carefor most cancers fall short in some or all of these criteria.

Cellular immunotherapy has been shown to result in specific tumorelimination and has the potential to provide specific and effectivecancer therapy (Ho, W. Y. et al. 2003. Cancer Cell 3:1318-1328; Morris,E. C. et al. 2003. Clin. Exp. Immunol. 131:1-7; Rosenberg, S. A. 2001.Nature 411:380-384; Boon, T. and P. van der Bruggen. 1996. J. Exp. Med.183:725-729). T cells have often been the effector cells of choice forcancer immunotherapy due to their selective recognition and powerfuleffector mechanisms. T cells recognize specific peptides derived frominternal cellular proteins in the context of self-majorhistocompatability complex (MHC) using their T cell receptors (TCR).

It is recognized in the art that the TCR complex associates in precisefashion by the formation of dimers and association of these dimers(TCR-alpha/beta, CD3-gamma/epsilon, CD3-delta/epsilon, and CD3-zetadimer) into one TCR complex that can be exported to the cell surface.The inability of any of these complexes to form properly will inhibitTCR assembly and expression (Call, M. E. et al., (2007) Nature Rev.Immunol., 7:841-850; Call, M. E. et al., (2005) Annu. Rev. Immunol.,23:101-125).

Particular amino acid residues in the respective TCR chains have beenidentified as important for proper dimer formation and TCR assembly. Inparticular, for TCR-alpha, these key amino acids in the transmembraneportion are arginine (for association with CD3-zeta) and lysine (forassociation with the CD3-epsilon/delta dimer). For TCR-beta, the keyamino acid in the transmembrane portion is a lysine (for associationwith CD3-epsilon/gamma dimer). For CD3-gamma, the key amino acid in thetransmembrane portion is a glutamic acid. For CD3-delta, the key aminoacid in the transmembrane portion is an aspartic acid. For CD3-epsilon,the key amino acid in the transmembrane portion is an aspartic acid. ForCD3-zeta, the key amino acid in the transmembrane portion is an asparticacid (Call, M. E. et al., (2007) Nature Rev. Immunol., 7:841-850; Call,M. E. et al., (2005) Annu. Rev. Immunol., 23:101-125).

Peptides derived from altered or mutated proteins in tumors can berecognized by specific TCRs. Several key studies have led to theidentification of antigens associated with specific tumors that havebeen able to induce effective cytotoxic T lymphocyte (CTL) responses inpatients (Ribas, A. et al. 2003. J. Clin. Oncol. 21:2415-2432). T celleffector mechanisms include the ability to kill tumor cells directly andthe production of cytokines that activate other host immune cells andchange the local tumor microenvironment. Theoretically, T cells couldidentify and destroy a tumor cell expressing a single mutated peptide.Adoptive immunotherapy with CTL clones specific for MART1 or gp100 withlow dose IL-2 has been effective in reduction or stabilization of tumorburden in some patients (Yee, C. et al. 2002. Proc. Natl. Acad. Sci. USA99:16168-16173). Other approaches use T cells with a defined anti-tumorreceptor. These approaches include genetically modifying CTLs with newantigen-specific T cell receptors that recognize tumor peptides and MHC,chimeric antigen receptors (CARS) derived from single chain antibodyfragments (scFv) coupled to an appropriate signaling element, or the useof chimeric NK cell receptors (Ho, W. Y. et al. 2003. Cancer Cell3:431-437; Eshhar, Z. et al. 1993. Proc. Natl. Acad. Sci. USA90:720-724; Maher, J. and E. T. Davies. 2004. Br. J. Cancer 91:817-821;Zhang, T. et al. 2005. Blood 106:1544-1551).

Cell-based therapies are used in patients who have failed conventionalchemotherapy or radiation treatments, or have relapsed, often havingattempted more than one type of therapy. The immune cells from patientswith advanced cancer, who may have gone through rounds of chemotherapy,do not respond as robustly as healthy individuals. Moreover, cancerpatients are often elderly and may suffer from other diseases that maylimit the potential of their immune cells to become primed effectorcells, even after in vitro activation and expansion. In addition, eachcancer patient must provide a sufficient number of their own immunecells in order for them to be engineered to express a new immunereceptor. Because each therapy must be custom made for the patient, thisprocess requires weeks from the time the decision to undertake suchtherapy is made; meanwhile, the cancer continues to grow. U.S. patentapplication publication no. US 2002/0039576 discloses a method formodulating T cell activity, where the T cells used have a phenotype ofCD3⁺-αβ-TcR⁺CD4⁻CD8⁻CD28⁻NK1.1⁻. U.S. patent application publication no.US 2006/0166314 discloses use of mutated T cells to treat cancer wherethe T cells are ones with a T cell response-mediating MDM2protein-specific αβ-T cell receptor.

Cancer is not the only disease wherein T cell manipulation could beeffective therapy. It is known that active T cell receptors on T cellsare critical to the response of the body to stimulate immune systemactivity. For example, it has been shown that T cell receptor diversityplays a role in graft-versus-host-disease (GVHD), in particular chronicGVHD (Anderson et al. (2004) Blood 104:1565-1573). In fact,administration of T cell receptor antibodies has been shown to reducethe symptoms of acute GVHD (Maeda et al. (2005) Blood 106:749-755).

There remains a need for more effective T cell-based therapies for thetreatment of certain diseases and disorders, and methods of treatmentbased on the design of new types of T cells.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, this invention broadly relates to isolated, modifiedT cells that do not express a functional T cell receptor (TCR). In thisembodiment, the T cells are TCR-deficient in the expression of afunctional TCR. In another embodiment of the invention, TCR-deficient Tcells are engineered to express a functional non-TCR receptor, such asfor example a chimeric receptor. These cells also function as a platformto allow the expression of other targeting receptors, receptors that maybe useful in specific diseases, while retaining the effector functionsof T cells, albeit without a functioning TCR.

The invention contemplates populations of TCR-deficient T cells, andcompositions comprising the same. The invention also contemplatesmethods of making said TCR-deficient T cells, and methods of reducing orameliorating, or preventing or treating, diseases and disorders usingsaid TCR-deficient T cells, populations thereof, or therapeuticcompositions comprising the same. In one embodiment, this compositioncan be used to treat cancer, infection, one or more autoimmunedisorders, radiation sickness, or to prevent or treat graft versus hostdisease (GVHD) or transplantation rejection in a subject undergoingtransplant surgery.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates chimeric NK receptors described herein. Extracellular(EC), transmembrane (TM), and cytoplasmic (Cyp) portions are indicated.Wild-type (WT) and chimeric (CH) forms of the receptors are indicated,wherein NH₂ denotes the N-terminus and COOH denotes the C-terminus.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

In the context of the present invention, by a “TCR-deficient T cell”, ora similar phrase is intended an isolated T cell(s) that lacks expressionof a functional TCR, is internally capable of inhibiting its own TCRproduction, and further wherein progeny of said T cell(s) may also bereasonably expected to be internally capable of inhibiting their own TCRproduction. Internal capability is important in the context of therapywhere TCR turnover timescales (˜hours) are much faster than demonstrableefficacy timescales (days-months), i.e., internal capability is requiredto maintain the desired phenotype during the therapeutic period. Thismay e.g., be accomplished by different means as described infra, e.g.,by engineering a T cell such that it does not express any functional TCRon its cell surface, or by engineering the T cell such that it does notexpress one or more of the subunits that comprise a functional TCR andtherefore does not produce a functional TCR or by engineering a T cellsuch that it produces very little functional TCR on its surface, orwhich expresses a substantially impaired TCR, e.g by engineering the Tcell to express mutated or truncated forms of one or more of thesubunits that comprise the TCR, thereby rendering the T cell incapableof expressing a functional TCR or resulting in a cell that expresses asubstantially impaired TCR. The different subunits that comprise afunctional TCR are described infra. Whether a cell expresses afunctional TCR may be determined using known assay methods such as areknown in the art described herein. By a “substantially impaired TCR”applicants mean that this TCR will not substantially elicit an adverseimmune reaction in a host, e.g., a GVHD reaction.

As described in detail infra, optionally these TCR-deficient cells maybe engineered to comprise other mutations or transgenes that e.g.,mutations or transgenes that affect T cell growth or proliferation,result in expression or absence of expression of a desired gene or geneconstruct, e.g., another receptor or a cytokine or otherimmunomodulatory or therapeutic polypeptide or a selectable marker suchas a dominant selectable marker gene, e.g., DHFR or neomycintransferase.

“Allogeneic T cell” refers to a T cell from a donor having a tissue HLAtype that matches the recipient. Typically, matching is performed on thebasis of variability at three or more loci of the HLA gene, and aperfect match at these loci is preferred. In some instances allogeneictransplant donors may be related (usually a closely HLA matchedsibling), syngeneic (a monozygotic ‘identical’ twin of the patient) orunrelated (donor who is not related and found to have very close degreeof HLA matching). The HLA genes fall in two categories (Type I and TypeII). In general, mismatches of the Type-I genes (i.e. HLA-A, HLA-B, orHLA-C) increase the risk of graft rejection. A mismatch of an HLA TypeII gene (i.e. HLA-DR, or HLA-DQB1) increases the risk ofgraft-versus-host disease.

In the context of the present invention, a “bank of tissue matchedTCR-deficient T cells” refers to different compositions each containingT cells of a specific HLA allotype which are rendered TCR-deficientaccording to the invention. Ideally this bank will comprise compositionscontaining T cells of a wide range of different HLA types that arerepresentative of the human population. Such a bank of engineeredTCR-deficient T cells will be useful as it will facilitate theavailability of T cells suitable for use in different recipients such ascancer patients.

In the context of the present invention, a “therapeutically effectiveamount” is identified by one of skill in the art as being an amount ofTCR-deficient T cells that, when administered to a patient, alleviatesthe signs and or symptoms of the disease (e.g., cancer, infection orGVHD). The actual amount to be administered can be determined based onstudies done either in vitro or in vivo where the functionalTCR-deficient T cells exhibit pharmacological activity against disease.For example, the functional TCR-deficient T cells may inhibit tumor cellgrowth either in vitro or in vivo and the amount of functionalTCR-deficient T cells that inhibits such growth is identified as atherapeutically effective amount.

A “pharmaceutical composition” refers to a chemical or biologicalcomposition suitable for administration to a mammal. Such compositionsmay be specifically formulated for administration via one or more of anumber of routes, including but not limited to buccal, intraarterial,intracardial, intracerebroventricular, intradermal, intramuscular,intraocular, intraperitoneal, intraspinal, intrathecal, intravenous,oral, parenteral, rectally via an enema or suppository, subcutaneous,subdermal, sublingual, transdermal, and transmucosal. In addition,administration can occur by means of injection, liquid, gel, drops, orother means of administration.

As used herein, a nucleic acid construct or nucleic acid sequence isintended to mean a DNA molecule which can be transformed or introducedinto a T cell and be transcribed and translated to produce a product(e.g., a chimeric receptor or a suicide protein).

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for asignal sequence is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading frame. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites oralternatively via a PCR/recombination method familiar to those skilledin the art (Gateway® Technology; Invitrogen, Carlsbad Calif.). If suchsites do not exist, the synthetic oligonucleotide adapters or linkersare used in accordance with conventional practice.

The invention contemplates compositions and methods for reducing orameliorating, or preventing or treating, diseases or conditions such ascancer, infectious disease, GVHD, transplantation rejection, one or moreautoimmune disorders, or radiation sickness. In a non-limitingembodiment, the compositions are based on the concept of providing anallogeneic source of isolated human T cells, namely TCR-deficient Tcells, that can be manufactured in advance of patient need andinexpensively. The ability to create a single therapeutic product at asingle site using processes that are well controlled is attractive interms of both cost and quality considerations. The change from anautologous to an allogeneic source for T cells offers significantadvantages. For example, it has been estimated that a single healthydonor could supply T cells sufficient to treat dozens of patients aftertransduction and expansion.

According to the present invention, modified allogeneic T cells areproduced that do not express functional T cell receptors (TCRs). It isto be understood that some, or even all, of the TCR subunits/dimers maybe expressed on the cell surface, but that the T cell does not expressenough functional TCR to induce an undesirable reaction in the host.Without functional TCRs on their surface, the allogeneic T cells fail tomount an undesired immune response to host cells. As a result, theseTCR-deficient T cells fail to cause GVHD, for example, as they cannotrecognize the host MHC molecules. Additionally, these TCR-deficient Tcells can be engineered to simultaneously express functional, non-TCR,disease-specific receptors.

As is well known to one of skill in the art, various methods are readilyavailable for isolating allogeneic T cells from a subject. For example,using cell surface marker expression or using commercially availablekits (e.g., ISOCELL™ from Pierce, Rockford, Ill.).

For cancer therapy, the approach encompasses producing an isolated poolof TCR-deficient T effector cells, e.g., of a desired tissue allotypethat do not express a functional form of their endogenous TCR or whichexpress substantially reduced levels of endogenous TCR compared to wildtype T cells such that they do not elicit an immune response uponadministration (such as GVHD), but instead express a functional, non-TCRreceptor that recognizes tumor cells, or express another polypeptidethat does not appreciably, or at all, attack non-disease associatedcells, e.g., normal (non-tumorigenic) cells that do not express theantigen or ligand recognized by the tumor specific receptor or whichexpress said antigen or ligand at reduced levels relative to tumorcells. It is understood by those skilled in the art that certaintumor-associated antigens are expressed in non-cancerous tissues, butthey are viable therapeutic targets in a tumor-bearing host. Withrespect thereto it is generally understood by those skilled in the artthat certain non-TCR, tumor-specific receptors are expressed innon-cancerous tissues, but are viable therapeutic targets in atumor-bearing host as they may be expressed at significantly reducedlevels in normal than tumor cells.

While not necessary for most therapeutic usages of the subjectTCR-deficient T cells, in some instances it may be desirable to removesome or all of the donor T cells from the host shortly after they havemediated their anti-tumor effect. This may be facilitated by engineeringthe T cells to express additional receptors or markers that facilitatetheir removal and/or identification in the host such as GFP and thelike. While the present invention should substantially eliminate anypossibility of GVHD or other adverse immune reaction in the recipientthis may be desired in some individuals. This should not compromiseefficacy as it has already been shown that donor T cells do not need toremain long in the host for a long-term anti-tumor effect to beinitiated (Zhang, T., et al. 2007. Cancer Res. 67:11029-11036; Barber,A. et al. 2008. J. Immunol. 180:72-78).

In one embodiment of the invention, nucleic acid constructs introducedinto engineered T cells further contains a suicide gene such asthymidine kinase (TK) of the HSV virus (herpesvirus) type I (Bonini, etal. (1997) Science 276:1719-1724), a Fas-based “artificial suicide gene”(Thomis, et al. (2001) Blood 97:1249-1257), or E. coli cytosinedeaminase gene which are activated by gancyclovir, AP1903, or5-fluorocytosine, respectively. The suicide gene is advantageouslyincluded in the nucleic acid construct of the present invention toprovide for the opportunity to ablate the transduced T cells in case oftoxicity and to destroy the chimeric construct once a tumor has beenreduced or eliminated. The use of suicide genes for eliminatingtransformed or transduced cells is well-known in the art. For example,Bonini, et al. ((1997) Science 276:1719-1724) teach that donorlymphocytes transduced with the HSV-TK suicide gene provide antitumoractivity in patients for up to one year and elimination of thetransduced cells is achieved using ganciclovir. Further, Gonzalez, etal. ((2004) J. Gene Med. 6:704-711) describe the targeting ofneuroblastoma with cytotoxic T lymphocyte clones genetically modified toexpress a chimeric scFvFc:zeta immunoreceptor specific for an epitope onL1-CAM, wherein the construct further expresses the hygromycin thymidinekinase (HyTK) suicide gene to eliminate the transgenic clones.

It is contemplated that the suicide gene can be expressed from the samepromoter as the shRNA, minigene, or non-TCR receptor, or from adifferent promoter. Generally, however, nucleic acid sequences encodingthe suicide protein and shRNA, minigene, or non-TCR receptor reside onthe same construct or vector. Expression of the suicide gene from thesame promoter as the shRNA, minigene, or non-TCR receptor can beaccomplished using any well-known internal ribosome entry site (IRES).Suitable IRES sequences which can be used in the nucleic acid constructof the present invention include, but are not limited to, IRES fromEMCV, c-myc, FGF-2, poliovirus and HTLV-1. By way of illustration only,a nucleic acid construct for expressing a chimeric receptor can have thefollowing structure: promoter->chimeric receptor->IRES->suicidal gene.Alternatively, the suicide gene can be expressed from a differentpromoter than that of the chimeric receptor (e.g., promoter 1->chimericreceptor->promoter 2->suicidal gene).

Because of the broad application of T cells for cell therapies, and theimproved nature of the T cells of the invention, the present inventionencompasses any method or composition wherein T cells aretherapeutically desirable. Such compositions and methods include thosefor reducing or ameliorating, or preventing or treating cancer, GVHD,transplantation rejection, infection, one or more autoimmune disorders,radiation sickness, or other diseases or conditions that are based onthe use of T cells derived from an allogeneic source that lackexpression of functional TCR.

As indicated, further embodiments of the invention embrace recombinantexpression of receptors in said TCR-deficient T cells, such as chimericNKG2D, chimeric Fv domains, NKG2D, or any other receptor to initiatesignals to T cells, thereby creating potent, specific effector T cells.One of skill in the art can select the appropriate receptor to beexpressed by the TCR-deficient T cell based on the disease to betreated. For example, receptors that can be expressed by theTCR-deficient T cell for treatment of cancer would include any receptorto a ligand that has been identified on cancer cells. Such receptorsinclude, but are not limited to, NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL,CD26, NKRP1, NKp30, NKp44, NKp46, CD244 (2B4), DNAM-1, and NKp80.

In another embodiment of the invention, such receptors include, but notlimited to, chimeric receptors comprising a ligand binding domainobtained from NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1,NKp30, NKp44, NKp46, CD244 (2B4), DNAM-1, and NKp80, or an anti-tumorantibody such as anti-Her2neu or anti-EGFR, and a signaling domainobtained from CD3-zeta, Dap10, CD28, 41BB, and CD40L. In one embodimentof the invention, the chimeric receptor binds MIC-A, MIC-B, Her2neu,EGFR, mesothelin, CD38, CD20, CD19, PSA, MUC1, MUC2, MUC3A, MUC3B, MUC4,MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13, MUC15, MUC16, MUC17,MUC19, MUC20, estrogen receptor, progesterone receptor, RON, or one ormore members of the ULBP/RAET1 family including ULBP1, ULBP2, ULBP3,ULBP4, ULBP5, and ULBP6.

In the methods of the present invention a patient suffering from cancer,GVHD, transplantation rejection, infection, one or more autoimmunedisorders, or radiation sickness is administered a therapeuticallyeffective amount of a composition comprising said TCR-deficient T cells.In another embodiment of the invention, a therapeutically effectiveamount of a composition comprising said TCR-deficient T cells isadministered to prevent, treat, or reduce GVHD, transplantationrejection, or cancer.

Methods of Producing TCR-Deficient T-Cells

T cells stably lacking expression of a functional TCR according to theinvention may be produced using a variety of approaches. T cellsinternalize, sort, and degrade the entire T cell receptor as a complex,with a half-life of about 10 hours in resting T cells and 3 hours instimulated T cells (von Essen, M. et al. 2004. J. Immunol. 173:384-393).Proper functioning of the TCR complex requires the proper stoichiometricratio of the proteins that compose the TCR complex. TCR function alsorequires two functioning TCR zeta proteins with ITAM motifs. Theactivation of the TCR upon engagement of its MHC-peptide ligand requiresthe engagement of several TCRs on the same T cell, which all must signalproperly. Thus, if a TCR complex is destabilized with proteins that donot associate properly or cannot signal optimally, the T cell will notbecome activated sufficiently to begin a cellular response.

In one embodiment of the invention, TCR expression is eliminated usingsmall-hairpin RNAs (shRNAs) that target nucleic acids encoding specificTCRs (e.g., TCR-α and TCR-β) and/or CD3 chains in primary T cells. Byblocking expression of one or more of these proteins, the T cell will nolonger produce one or more of the key components of the TCR complex,thereby destabilizing the TCR complex and preventing cell surfaceexpression of a functional TCR. Even though some TCR complexes can berecycled to the cell surface, the shRNA will prevent new production ofTCR proteins resulting in degradation and removal of the entire TCRcomplex, resulting in the production of a T cell having a stabledeficiency in functional TCR expression.

Expression of shRNAs in primary T cells can be achieved using anyconventional expression system, e.g., a lentiviral expression system.Although lentiviruses are useful for targeting resting primary T cells,not all T cells will express the shRNAs. Some of these T cells may notexpress sufficient amounts of the shRNAs to allow enough inhibition ofTCR expression to alter the functional activity of the T cell. Thus, Tcells that retain moderate to high TCR expression after viraltransduction can be removed, e.g., by cell sorting or separationtechniques, so that the remaining T cells are deficient in cell surfaceTCR or CD3, enabling the expansion of an isolated population of T cellsdeficient in expression of functional TCR or CD3.

In a non-limiting embodiment of the invention, exemplary targetingshRNAs have been designed for key components of the TCR complex as setforth below (Table 1).

TABLE 1 Target SE

 ID Target base shRNA Sequence GC%

O: TCR-β 18^(a) AGTGCGAGGAGATTCGGCAGCTTAT 52  1 21^(a)GCGAGGAGATTCGGCAGCTTATTTC 52  2 48^(a) CCACCATCCTCTATGAGATCTTGCT 48  354^(a) TCCTCTATGAGATCTTGCTAGGGAA 44  4 TCR-α  3^(b)TCTATGGCTTCAACTGGCTAGGGTG 52  5 76^(b) CAGGTAGAGGCCTTGTCCACCTAAT 52  601^(b) GCAGCAGACACTGCTTCTTACTTCT 48  7 07^(b) GACACTGCTTCTTACTTCTGTGCTA44  8 CD3-ϵ 89^(c) CCTCTGCCTCTTATCAGTTGGCGTT 52  9 27^(c)GAGCAAAGTGGTTATTATGTCTGCT 40 10 62^(c) AAGCAAACCAGAAGATGCGAACTTT 40 1145 GACCTGTATTCTGGCCTGAATCAGA 48 12 GGCCTCTGCCTCTTATCAGTT 52 13GCCTCTGCCTCTTATCAGTTG 52 14 GCCTCTTATCAGTTGGCGTTT 48 15AGGATCACCTGTCACTGAAGG 52 16 GGATCACCTGTCACTGAAGGA 52 17GAATTGGAGCAAAGTGGTTAT 38 18 GGAGCAAAGTGGTTATTATGT 38 19GCAAACCAGAAGATGCGAACT 48 20 ACCTGTATTCTGGCCTGAATC 48 21GCCTGAATCAGAGACGCATCT 52 22 CTGAAATACTATGGCAACACAATGATAAA 31 23AAACATAGGCAGTGATGAGGATCACCTGT 45 24 ATTGTCATAGTGGACATCTGCATCACTGG 45 25CD3-δ^(d) CTGTATTCTGGCCTGAATCAGAGACGCAT 48 26 GATACCTATAGAGGAACTTGA 3827 GACAGAGTGTTTGTGAATTGC 43 28 GAACACTGCTCTCAGACATTA 43 29GGACCCACGAGGAATATATAG 48 30 GGTGTAATGGGACAGATATAT 38 31GCAAGTTCATTATCGAATGTG 38 32 GGCTGGCATCATTGTCACTGA 52 33GCTGGCATCATTGTCACTGAT 48 34 GCATCATTGTCACTGATGTCA 43 35GCTTTGGGAGTCTTCTGCTTT 48 36 TGGAACATAGCACGTTTCTCTCTGGCCTG 52 37CTGCTCTCAGACATTACAAGACTGGACCT 48 38 ACCGTGGCTGGCATCATTGTCACTGATGT 52 39TGATGCTCAGTACAGCCACCTTGGAGGAA 52 40 GGCTATCATTCTTCTTCAAGG 43 41GCCCAGTCAATCAAAGGAAAC 48 42 GGTTAAGGTGTATGACTATCA 38 43GGTTCGGTACTTCTGACTTGT 48 44 CD3-γ^(e) GAATGTGTCAGAACTGCATTG 43 45GCAGCCACCATATCTGGCTTT 52 46 GGCTTTCTCTTTGCTGAAATC 43 47GCTTTCTCTTTGCTGAAATCG 43 48 GCCACCTTCAAGGAAACCAGT 52 49GAAACCAGTTGAGGAGGAATT 43 50 GGCTTTCTCTTTGCTGAAATCGTCAGCAT 45 51AGGATGGAGTTCGCCAGTCGAGAGCTTCA 55 52 CCTCAAGGATCGAGAAGATGACCAGTACA 48 53TACAGCCACCTTCAAGGAAACCAGTTGAG 48 54 ^(a)With reference to Accession No.EU030678. ^(b)With reference to Accession No. AY247834. ^(c)Withreference to Accession No. NM_000733. ^(d)With reference to AccessionNo. NM_000732. ^(e)With reference to Accession No. NM_000073.

TCR-alpha, TCR-beta, TCR-gamma, TCR-delta, CD3-gamma, CD3-delta,CD3-epsilon, or CD3-zeta mRNAs can be targeted separately or togetherusing a variety of targeting shRNAs. The TCR-β and TCR-α chains arecomposed of variable and constant portions. Several targeting shRNAshave been designed for the constant portions of these TCR/CD3 sequences.One or a combination of shRNAs can be used for each molecular target toidentify the most efficient inhibitor of TCR expression. Usingestablished protocols, each shRNA construct is cloned into, e.g., apLko. 1 plasmid, with expression controlled by a promoter routinely usedin the art, e.g., the U6p promoter. The resulting construct can bescreened and confirmed for accuracy by sequencing. The shRNA expressionplasmid can then be transfected into any suitable host cell (e.g.,293T), together with a packaging plasmid and an envelope plasmid forpackaging. Primary human peripheral blood mononuclear cells (PBMCs) areisolated from healthy donors and activated with low dose solubleanti-CD3 and 25 U/ml rhulL-2 for 48 hours. Although it is not requiredto activate T cells for lentiviral transduction, transduction works moreefficiently and allows the cells to continue to expand in IL-2. Theactivated cells are washed and transduced, e.g., using a 1 hourspin-fection at 30° C., followed by a 7 hour resting period.

In another embodiment of the invention, over-expression of adominant-negative inhibitor protein is capable of interrupting TCRexpression or function. In this embodiment of the invention, a minigenethat incorporates part, or all, of a polynucleotide encoding for one ofthe TCR components (e.g., TCR-alpha, TCR-beta, CD3-gamma, CD3-delta,CD3-epsilon, or CD3-zeta) is prepared, but is modified so that: (1) itlacks key signaling motifs (e.g. an ITAM) required for protein function;(2) is modified so it does not associate properly with its other naturalTCR components; or (3) can associate properly but does not bind ligands(e.g. a truncated TCR beta minigene).

These minigenes may also encode a portion of a protein that serves as ameans to identify the over-expressed minigene. For example,polynucleotides encoding a truncated CD19 protein, which contains thebinding site for anti-CD19 mAbs, can be operably linked to the minigeneso that the resulting cell that expresses the minigene will express theencoded protein and can be identified with anti-CD19 mAbs. Thisidentification enables one to determine the extent of minigeneexpression and isolate cells expressing this protein (and thus lack afunctional TCR).

In one embodiment of the invention, over-expression of a minigenelacking a signaling motif(s) lead to a TCR complex that cannot signalproperly when the TCR is engaged by its MHC-peptide ligand on anopposing cell. In a non-limiting embodiment of the invention, highexpression of this minigene (and the encoded polypeptide) outcompetesthe natural complete protein when the TCR components associate,resulting in a TCR complex that cannot signal. In another embodiment ofthe invention, the minigene comprises, or alternatively consists of, apolynucleotide encoding full or partial CD3-zeta, CD3-gamma, CD3-delta,or CD3-epsilon polypeptides lacking the ITAM motifs required forsignaling. The CD3-zeta protein contains three ITAM motifs in thecytoplasmic portion, and in one embodiment of the invention, removal ofall of these through truncation inhibits proper TCR signaling in anycomplexes where this modified protein is incorporated. The construct mayincorporate ITIM or other signaling motifs, which are known to altercell signaling and promote inhibitory signals through the recruitment ofphosphatases such as SHP1 and SHP2.

In another embodiment of the invention, over-expression of a minigene ismodified so that the encoded polypeptide can associate with some, butnot all, of its natural partners, creating competition with the normalprotein for those associating proteins. In another non-limitinghypothesis of the invention, high level expression of the minigene (andthe encoded polypeptide) outcompetes the natural partner proteins andprevents assembly of a functional TCR complex, which requires allcomponents to associate in the proper ratios and protein-proteininteractions. In another embodiment of the invention, minigenescomprise, or alternatively consist of, all or part of thepolynucleotides encoding full-length proteins (e.g., TCR-alpha,TCR-beta, CD3-gamma, CD3-delta, CD3-epsilon, or CD3-zeta), butcontaining selected deletions in the sequence coding for amino acids inthe transmembrane portion of the protein that are known to be requiredfor assembly with other TCR/CD3 proteins.

In a preferred embodiment of the invention, selected deletions in thesequence coding for amino acids in the transmembrane portion of theprotein known to be required for assembly with other TCR/CD3 proteinsinclude, but are not limited to: the arginine residue at position 5 inthe TCR-alpha transmembrane region; the lysine residue at position 10 inthe TCR-alpha transmembrane region; the lysine residue at position 9 inthe TCR-beta transmembrane region; the glutamic acid residue in thetransmembrane region of CD3-gamma; the aspartic acid residue in thetransmembrane region of CD3-delta-epsilon; the aspartic acid residue inthe transmembrane region of CD3-epsilon; and the aspartic acid residuein the transmembrane region of CD3-zeta.

Over-expression of a truncated TCR-alpha, TCR-beta, TCR-gamma, orTCR-delta protein results in a TCR complex that cannot bind toMHC-peptide ligands, and thus will not function to activate the T cell.In another embodiment of the invention, minigenes comprise, oralternatively consist of, polynucleotides encoding the entirecytoplasmic and transmembrane portions of these proteins and portions ofthe extracellular region, but lacks polynucleotides encoding all or partof the first extracellular domain (i.e., the most outer domaincontaining the ligand binding site). In a preferred embodiment, saidminigene polynucleotides do not encode Valpha and Vbeta polypeptides ofthe TCR-alpha and TCR-beta chains. In one embodiment, the minigenepolynucleotides may be operably linked to polynucleotides encoding aprotein epitope tag (e.g. CD19), thereby allowing mAb identification ofcells expressing these genes.

In another embodiment, these minigenes can be expressed using a strongviral promoter, such as the 5′LTR of a retrovirus, or a CMV or SV40promoter. Typically, this promoter is immediately upstream of theminigene and leads to a high expression of the minigene mRNA. In anotherembodiment, the construct encodes a second polynucleotide sequence underthe same promoter (using for example an IRES DNA sequence between) oranother promoter. This second polynucleotide sequence may encode for afunctional non-TCR receptor providing specificity for the T cell.Examples of this polynucleotide include, but are not limited to,chimeric NKG2D, chimeric NKp30, chimeric NKp46, or chimericanti-Her2neu. In a further embodiment, promoter-minigenes areconstructed into a retroviral or other suitable expression plasmid andtransfected or transduced directly into T cells using standard methods(Zhang, T. et al., (2006) Cancer Res., 66(11) 5927-5933; Barber, A. etal., (2007) Cancer Res., 67(10):5003-5008).

After viral transduction and expansion using any of the methodsdiscussed previously, any T cells that still express TCR/CD3 are removedusing anti-CD3 mAbs and magnetic beads using Miltenyi selection columnsas previously described (Barber, A. et al., (2007) Cancer Res.,67(10):5003-5008). The T cells are subsequently washed and cultured inIL-2 (25 U/ml) for 3 to 7 days to allow expansion of the effector cellsin a similar manner as for use of the cells in vivo.

The expression of TCR αβ and CD3 can be evaluated by flow cytometry andquantitative real-time PCR (QRT-PCR). Expression of TCR-α, TCR-β, CD3ε,CD3-ζ, and GAPDH (as a control) mRNA can be analyzed by QRT-PCR using anABI7300 real-time PCR instrument and gene-specific TAQMAN® primers usingmethods similar to those used in Sentman, C. L. et al. ((2004) J.Immunol. 173:6760-6766). Changes in cell surface expression can bedetermined using antibodies specific for TCR-α, TCR-β, CD3ε, CD8, CD4,CD5, and CD45.

It is possible that a single shRNA species may not sufficiently inhibitTCR expression on the cell surface. In this case, multiple TCR shRNAsmay be used simultaneously to target multiple components of the TCRcomplex. Each component is required for TCR complex assembly at the cellsurface, so a loss of one of these proteins can result in loss of TCRexpression at the cell surface. While some or even all TCR expressionmay remain, it is the receptor function which determines whether thereceptor induces an immune response. The functional deficiency, ratherthan complete cell surface absence, is the critical measure. In general,the lower the TCR expression, the less likely sufficient TCRcross-linking can occur to lead to T cell activation via the TCRcomplex. While particular embodiments embrace the targeting ofTCR-alpha, TCR-beta, and CD3-epsilon, other components of the TCRcomplex, such as CD3-gamma, CD3-delta, or CD3-zeta, can also betargeted.

The primary aim of removing the TCR from the cell surface is to preventthe activation of the T cell to incompatible MHC alleles. To determinewhether the reduction in TCR expression with each shRNA or minigeneconstruct is sufficient to alter T cell function, the T cells can betested for: (1) cell survival in vitro; (2) proliferation in thepresence of mitomycin C-treated allogeneic PBMCs; and (3) cytokineproduction in response to allogeneic PBMCs, anti-CD3 mAbs, or anti-TCRmAbs.

To test cell survival, transduced T cells are propagated in completeRPMI medium with rhulL-2 (25 U/ml). Cells are plated at similardensities at the start of culture and a sample may be removed for cellcounting and viability daily for 7 or more days. To determine whetherthe T cells express sufficient TCR to induce a response againstallogeneic cells, transduced or control T cells are cultured withmitomycin C-treated allogeneic or syngeneic PBMCs. The T cells arepreloaded with CFSE, which is a cell permeable dye that divides equallybetween daughter cells after division. The extent of cell division canbe readily determined by flow cytometry. Another hallmark of T cellactivation is production of cytokines. To determine whether each shRNAconstruct inhibits T cell function, transduced T cells are cultured withanti-CD3 mAbs (25 ng/ml). After 24 hours, cell-free supernatants arecollected and the amount of IL-2 and IFN-γ produced is quantified byELISA. PMA/ionomycin are used as a positive control to stimulate the Tcells and T cells alone are used as a negative control.

It is possible that removal of TCR-alpha or TCR-beta components mayallow the preferential expansion of TCR-gamma/delta T cells. These Tcells are quite rare in the blood, however the presence of these cellscan be determined with anti-TCR-gamma/delta antibodies. If there is anoutgrowth of these cells, the targeting of CD3-epsilon, which isrequired for cell surface expression of both TCR-alpha/beta andTCR-gamma/delta at the cell surface, can be used. Both IL-2 and IFN-γare key effector cytokines that drive T cell expansion and macrophageactivation. Therefore, lack of production of these cytokines is a signof functional inactivation. It is also possible to measure changes inother cytokines, such as TNF-α. Any reduction in T cell survival uponelimination of TCR expression can be determined by culturing theTCR-deficient T cells with PBMCs, which better reflects the in vivoenvironment and provides support for T cell survival.

In another embodiment of the invention, The T cells stably deficient infunctional TCR expression express a functional, non-TCR receptor. Inthis embodiment, the removal of TCR function (as described previously)is further combined with expression of one or more exogenous non-TCRtargeting receptors (such as for example chimeric NKG2D (chNKG2D) or Fvmolecules). This embodiment provides “universal” cell products, whichcan be stored for future therapy of any patient with any type of cancer,provided a suitable targeting receptor is employed.

Further embodiments of the invention embrace recombinant expression ofreceptors in said TCR-deficient T cells, such as chNKG2D, chimeric Fvdomains, NKG2D, or any other receptor to initiate signals to T cells,thereby creating potent, specific effector T cells. One of skill in theart can select the appropriate receptor to be expressed by theTCR-deficient T cell based on the disease to be treated. For example,receptors that can be expressed by the TCR-deficient T cell fortreatment of cancer would include any receptor to a ligand that has beenidentified on cancer cells. Such receptors include, but are not limitedto, NKG2D (GENBANK accession number BC039836), NKG2A (GENBANK accessionnumber AF461812), NKG2C (GENBANK accession number AJ001684), NKG2F,LLT1, AICL, CD26, NKRP1, NKp30 (e.g., GENBANK accession numberAB055881), NKp44 (e.g., GENBANK accession number AJ225109), NKp46 (e.g.,GENBANK accession number AJ001383), CD244 (2B4), DNAM-1, and NKp80.

In another embodiment of the invention, such receptors include, but notlimited to, chimeric receptors comprising a ligand binding domainobtained from NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1,NKp30, NKp44, NKp46, CD244 (2B4), DNAM-1, and NKp80, or an anti-tumorantibody, such as anti-Her2neu and anti-EGFR, and a signaling domainobtained from CD3-zeta (CD3) (e.g., GENBANK accession number humanNM_198053), Dap10 (e.g., GENBANK accession number AF072845), CD28, 41BB,and/or CD40L.

In a further embodiment of the invention, the chimeric receptor bindsMIC-A, MIC-B, Her2neu, EGFR, mesothelin, CD38, CD20, CD19, PSA, MUC1,MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13,MUC15, MUC16, MUC17, MUC19, MUC20, estrogen receptor, progesteronereceptor, RON, or one or more members of the ULBP/RAET1 family includingULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

By way of illustration only, shRNAs or minigenes shown to eliminate cellsurface expression of the TCR complex are co-expressed with the chNKG2Dreceptor via one or more viral vectors. To achieve co-expression in onevector, the shRNA can be driven by a U6 promoter and the chNKG2Dreceptor by a PGK promoter. In another embodiment, if an IRES sequenceis used to separate the genetic elements then only one promoter is used.

A C-type lectin-like NK cell receptor protein particularly suitable foruse in the chimeric receptor includes a receptor expressed on thesurface of natural killer cells, wherein upon binding to its cognateligand(s) it alters NK cell activation. The receptor can work alone orin concert with other molecules. Ligands for these receptors aregenerally expressed on the surface of one or more tumor cell types,e.g., tumors associated with cancers of the colon, lung, breast, kidney,ovary, cervix, and prostate; melanomas; myelomas; leukemias; andlymphomas (Wu, et al. (2004) J. Clin. Invest. 114:60-568; Groh, et al.(1999) Proc. Natl. Acad. Sci. USA 96:6879-6884; Pende, et al. (2001)Eur. J. Immunol. 31:1076-1086) and are not widely expressed on thesurface of cells of normal tissues.

Examples of such ligands include, but are not limited to, MIC-A, MIC-B,heat shock proteins, ULBP binding proteins (e.g., ULPBs 1-4), andnon-classical HLA molecules such as HLA-E and HLA-G, whereas classicalMHC molecules such as HLA-A, HLA-B, or HLA-C and alleles thereof are notgenerally considered strong ligands of the C-type lectin-like NK cellreceptor protein of the present invention. C-type lectin-like NK cellreceptors which bind these ligands generally have a type II proteinstructure, wherein the N-terminal end of the protein is intracellular.In addition to any NK cell receptors previously listed above, furtherexemplary NK cell receptors of this type include, but are not limitedto, Dectin-1 (GENBANK accession number AJ312373 or AJ312372), Mast cellfunction-associated antigen (GENBANK accession number AF097358),HNKR-P1A (GENBANK accession number U11276), LLT1 (GENBANK accessionnumber AF133299), CD69 (GENBANK accession number NM_001781), CD69homolog, CD72 (GENBANK accession number NM_001782), CD94 (GENBANKaccession number NM_002262 or NM_007334), KLRF1 (GENBANK accessionnumber NM_016523), Oxidised LDL receptor (GENBANK accession numberNM_002543), CLEC-1, CLEC-2 (GENBANK accession number NM_016509), NKG2D(GENBANK accession number BC039836), NKG2C (GENBANK accession numberAJ001684), NKG2A (GENBANK accession number AF461812), NKG2E (GENBANKaccession number AF461157), WUGSC:H_DJ0701016.2, or MyeloidDAP12-associating lectin (MDL-1; GENBANK accession number AJ271684). Ina preferred embodiment of the invention, the NK cell receptor is humanNKG2D (SEQ ID NO:58) or human NKG2C (SEQ ID NO:59).

Similar type I receptors which would be useful in the chimeric receptorinclude NKp46 (GENBANK accession number AJ001383), NKp30 (GENBANKaccession number AB055881), or NKp44 (GENBANK accession numberAJ225109).

As an alternative to the C-type lectin-like NK cell receptor protein, aprotein associated with a C-type lectin-like NK cell receptor proteincan be used in the chimeric receptor protein. In general, proteinsassociated with C-type lectin-like NK cell receptor are defined asproteins that interact with the receptor and transduce signalstherefrom. Suitable human proteins which function in this manner furtherinclude, but are not limited to, DAP10 (e.g., GENBANK accession numberAF072845)(SEQ ID NO:60), DAP12 (e.g., GENBANK accession numberAF019562)(SEQ ID NO:61) and FcR gamma.

To the N-terminus of the C-type lectin-like NK cell receptor is fused animmune signaling receptor having an immunoreceptor tyrosine-basedactivation motif (ITAM),(Asp/Glu)-Xaa-Xaa-Tyr*-Xaa-Xaa-(Ile/Leu)-Xaa₆₋₈-Tyr*-Xaa-Xaa-(Ile/Leu)(SEQ ID NOS: 55-57) which is involved in the activation of cellularresponses via immune receptors. Similarly, when employing a proteinassociated with a C-type lectin-like NK cell receptor, an immunesignaling receptor can be fused to the C-terminus of said protein (FIG.1). Suitable immune signaling receptors for use in the chimeric receptorof the present invention include, but are not limited to, the zeta chainof the T-cell receptor, the eta chain which differs from the zeta chainonly in its most C-terminal exon as a result of alternative splicing ofthe zeta mRNA, the delta, gamma and epsilon chains of the T-cellreceptor (CD3 chains) and the gamma subunit of the FcR1 receptor. Inparticular embodiments, in addition to immune signaling receptorsidentified previously, the immune signaling receptor is CD3-zeta (CD3ζ)(e.g., GENBANK accession number human NM_198053)(SEQ ID NO:62), or humanFc epsilon receptor-gamma chain (e.g., GENBANK accession numberM33195)(SEQ ID NO:63) or the cytoplasmic domain or a splicing variantthereof.

In particular embodiments, a chimeric receptor of the present inventionis a fusion between NKG2D and CD3-zeta, or Dap10 and CD3-zeta.

In the nucleic acid construct of the present invention, the promoter isoperably linked to the nucleic acid sequence encoding the chimericreceptor of the present invention, i.e., they are positioned so as topromote transcription of the messenger RNA from the DNA encoding thechimeric receptor. The promoter can be of genomic origin orsynthetically generated. A variety of promoters for use in T cells arewell-known in the art (e.g., the CD4 promoter disclosed by Marodon, etal. (2003) Blood 101(9):3416-23). The promoter can be constitutive orinducible, where induction is associated with the specific cell type ora specific level of maturation. Alternatively, a number of well-knownviral promoters are also suitable. Promoters of interest include theβ-actin promoter, SV40 early and late promoters, immunoglobulinpromoter, human cytomegalovirus promoter, retrovirus promoter, and theFriend spleen focus-forming virus promoter. The promoters may or may notbe associated with enhancers, wherein the enhancers may be naturallyassociated with the particular promoter or associated with a differentpromoter.

The sequence of the open reading frame encoding the chimeric receptorcan be obtained from a genomic DNA source, a eDNA source, or can besynthesized (e.g., via PCR), or combinations thereof. Depending upon thesize of the genomic DNA and the number of introns, it may be desirableto use cDNA or a combination thereof as it is found that intronsstabilize the mRNA or provide T cell-specific expression (Barthel andGoldfeld (2003) J. Immunol. 171(7):3612-9). Also, it may be furtheradvantageous to use endogenous or exogenous non-coding regions tostabilize the mRNA.

For expression of a chimeric receptor of the present invention, thenaturally occurring or endogenous transcriptional initiation region ofthe nucleic acid sequence encoding N-terminal component of the chimericreceptor can be used to generate the chimeric receptor in the targethost. Alternatively, an exogenous transcriptional initiation region canbe used which allows for constitutive or inducible expression, whereinexpression can be controlled depending upon the target host, the levelof expression desired, the nature of the target host, and the like.

Likewise, the signal sequence directing the chimeric receptor to thesurface membrane can be the endogenous signal sequence of N-terminalcomponent of the chimeric receptor. Optionally, in some instances, itmay be desirable to exchange this sequence for a different signalsequence. However, the signal sequence selected should be compatiblewith the secretory pathway of T cells so that the chimeric receptor ispresented on the surface of the T cell.

Similarly, a termination region can be provided by the naturallyoccurring or endogenous transcriptional termination region of thenucleic acid sequence encoding the C-terminal component of the chimericreceptor. Alternatively, the termination region can be derived from adifferent source. For the most part, the source of the terminationregion is generally not considered to be critical to the expression of arecombinant protein and a wide variety of termination regions can beemployed without adversely affecting expression.

As will be appreciated by one of skill in the art, in some instances, afew amino acids at the ends of the C-type lectin-like natural killercell receptor (or protein associated therewith) or immune signalingreceptor can be deleted, usually not more than 10, more usually not morethan 5 residues. Also, it may be desirable to introduce a small numberof amino acids at the borders, usually not more than 10, more usuallynot more than 5 residues. The deletion or insertion of amino acids willusually be as a result of the needs of the construction, providing forconvenient restriction sites, ease of manipulation, improvement inlevels of expression, or the like. In addition, the substitute of one ormore amino acids with a different amino acid can occur for similarreasons, usually not substituting more than about five amino acids inany one domain.

The chimeric construct, which encodes the chimeric receptor can beprepared in conventional ways. Since, for the most part, naturalsequences are employed, the natural genes are isolated and manipulated,as appropriate (e.g., when employing a Type II receptor, the immunesignaling receptor component may have to be inverted), so as to allowfor the proper joining of the various components. Thus, the nucleic acidsequences encoding for the N-terminal and C-terminal proteins of thechimeric receptor can be isolated by employing the polymerase chainreaction (PCR), using appropriate primers which result in deletion ofthe undesired portions of the gene. Alternatively, restriction digestsof cloned genes can be used to generate the chimeric construct. Ineither case, the sequences can be selected to provide for restrictionsites which are blunt-ended, or have complementary overlaps.

The various manipulations for preparing the chimeric construct can becarried out in vitro and in particular embodiments the chimericconstruct is introduced into vectors for cloning and expression in anappropriate host using standard transformation or transfection methods.Thus, after each manipulation, the resulting construct from joining ofthe DNA sequences is cloned, the vector isolated, and the sequencescreened to insure that the sequence encodes the desired chimericreceptor. The sequence can be screened by restriction analysis,sequencing, or the like.

It is contemplated that the chimeric construct can be introduced into Tcells as naked DNA or in a suitable vector. Methods of stablytransfecting T cells by electroporation using naked DNA are known in theart. See, e.g., U.S. Pat. No. 6,410,319. Naked DNA generally refers tothe DNA encoding a chimeric receptor of the present invention containedin a plasmid expression vector in proper orientation for expression.Advantageously, the use of naked DNA reduces the time required toproduce T cells expressing the chimeric receptor of the presentinvention.

Alternatively, a viral vector (e.g., a retroviral vector, adenoviralvector, adeno-associated viral vector, or lentiviral vector) can be usedto introduce the chimeric construct into T cells. Suitable vectors foruse in accordance with the method of the present invention arenon-replicating in the subject's T cells. A large number of vectors areknown which are based on viruses, where the copy number of the virusmaintained in the cell is low enough to maintain the viability of thecell. Illustrative vectors include the pFB-neo vectors (STRATAGENE™) aswell as vectors based on HIV, SV40, EBV, HSV or BPV. Once it isestablished that the transfected or transduced T cell is capable ofexpressing the chimeric receptor as a surface membrane protein with thedesired regulation and at a desired level, it can be determined whetherthe chimeric receptor is functional in the host cell to provide for thedesired signal induction (e.g., production of Rantes, Mip1-alpha, GM-CSFupon stimulation with the appropriate ligand).

As described above, primary human PBMCs are isolated from healthy donorsand activated with low-dose soluble anti-CD3 and rhulL-2,anti-CD3/anti-CD28 beads and rhulL-2, or irradiated antigen presentingcells and rhulL-2. Although it is not required to activate T cells forlentiviral transduction, transduction is more efficient and the cellscontinue to expand in IL-2. The activated cells are washed andtransduced as described herein, followed by a resting period. The cellsare washed and cultured in IL-2 for 3 to 7 days to allow expansion ofthe effector cells in a similar manner as for use of the cells in vivo.

The expression of TCRαβ, CD3, and NKG2D can be evaluated by flowcytometry and quantitative QRT-PCR as discussed herein. The number ofCD4+ and CD8+ T cells can also be determined. Overall cell numbers andthe percentage of TCR complex-deficient, TCR-competent, andchNKG2D-expressing T cells can be determined by flow cytometry. Thesenumbers can be compared to PBMCs that have been transduced with theshRNA or chNKG2D genes alone (as controls). Vector-only transduced cellscan also be included as controls.

After viral transduction and expansion, the TCR+ and TCR− cells can beseparated by mAbs with magnetic beads over Miltenyi columns andTCR-deficient T cells expressing the chNKG2D receptor are identified andisolated. For example, chNKG2D expression can be verified by QRT-PCRusing specific primers for the chNKG2D receptor (Zhang, T. et al. (2007)Cancer Res. 67:11029-11036; Barber, A. et al. (2008) J. Immunol.180:72-78). Function of these TCR-deficient chNKG2D+ cells can bedetermined by culturing the cells with allogeneic PBMCs or tumor cellsthat express NKG2D ligands. T cell proliferation and cytokine production(IFN-γ and IL-2) can be determined by flow cytometry and ELISA,respectively. To determine whether the T cells that have lost TCRfunction and retained chNKG2D function, transduced or control T cellswill be cultured with anti-CD3 (25 ng/ml) or mitomycin C-treatedallogeneic PBMCs, or syngeneic PBMCs. The extent of cytokine production(IFN-γ and IL-2) can be determined by ELISA. The T cells can bepreloaded with CFSE, which is a cell permeable dye that divides equallybetween daughter cells after division. The extent of cell division canbe readily determined by flow cytometry.

Another hallmark of T cell activation is production of cytokines. Todetermine whether TCR-deficient chNKG2D+ cells induce T cell activation,the T cells are cocultured with mitomycin C-treated allogeneic PBMCs,syngeneic PBMCs, or tumor cells: P815-MICA (a murine tumor expressinghuman MICA, a ligand for NKG2D), P815, A2008 (a human ovarian tumorcell, NKG2D ligand+), and U266 (a human myeloma cell line, NKG2Dligand+). After 24 hours, cell-free supernatants are collected and theamount of IL-2 and IFN-γ produced is quantified by ELISA. T cells aloneand culture with syngeneic PBMCs are used as a negative control.

Subsequently, the transduced T cells are reintroduced or administered tothe subject to activate anti-tumor responses in said subject. Tofacilitate administration, the transduced T cells according to theinvention can be made into a pharmaceutical composition or made implantappropriate for administration in vivo, with appropriate carriers ordiluents, which further can be pharmaceutically acceptable. The means ofmaking such a composition or an implant have been described in the art(see, for instance, Remington's Pharmaceutical Sciences, 16th Ed., Mack,ed. (1980)). Where appropriate, the transduced T cells can be formulatedinto a preparation in semisolid or liquid form, such as a capsule,solution, injection, inhalant, or aerosol, in the usual ways for theirrespective route of administration. Means known in the art can beutilized to prevent or minimize release and absorption of thecomposition until it reaches the target tissue or organ, or to ensuretimed-release of the composition. Desirably, however, a pharmaceuticallyacceptable form is employed which does not ineffectuate the cellsexpressing the chimeric receptor. Thus, desirably the transduced T cellscan be made into a pharmaceutical composition containing a balanced saltsolution, preferably Hanks' balanced salt solution, or normal saline.

Methods of Ameliorating or Reducing Symptoms of or Treating, orPreventing, Diseases and Disorders Using TCR-Deficient T-Cells

The invention is also directed to methods of reducing or ameliorating,or preventing or treating, diseases and disorders using theTCR-deficient T cells described herein, isolated populations thereof, ortherapeutic compositions comprising the same. In one embodiment, theTCR-deficient T cells described herein, isolated populations thereof, ortherapeutic compositions comprising the same are used to reduce orameliorate, or prevent or treat, cancer, infection, one or moreautoimmune disorders, radiation sickness, or to prevent or treat graftversus host disease (GVHD) or transplantation rejection in a subjectundergoing transplant surgery.

The TCR-deficient T cells described herein, isolated populationsthereof, or therapeutic compositions comprising the same are useful inaltering autoimmune or transplant rejection because these effector cellscan be grown in TGF-β during development and will differentiate tobecome induced T regulatory cells. In one embodiment, the functionalnon-TCR is used to give these induced T regulatory cells the functionalspecificity that is required for them to perform their inhibitoryfunction at the tissue site of disease. Thus, a large number ofantigen-specific regulatory T cells are grown for use in patients. Theexpression of FoxP3, which is essential for T regulatory celldifferentiation, can be analyzed by flow cytometry, and functionalinhibition of T cell proliferation by these T regulatory cells can beanalyzed by examining decreases in T cell proliferation after anti-CD3stimulation upon co-culture.

Another embodiment of the invention is directed to the use ofTCR-deficient T cells described herein, isolated populations thereof, ortherapeutic compositions comprising the same for the prevention ortreatment of radiation sickness. One challenge after radiation treatmentor exposure (e.g. dirty bomb exposure, radiation leak) or othercondition that ablates bone marrow cells (certain drug therapies) is toreconstitute the hematopoietic system. In patients undergoing a bonemarrow transplant, the absolute lymphocyte count on day 15post-transplant is correlated with successful outcome. Those patientswith a high lymphocyte count reconstitute well, so it is important tohave a good lymphocyte reconstitution. The reason for this effect isunclear, but it may be due to lymphocyte protection from infectionand/or production of growth factors that favors hematopoieticreconstitution.

In this embodiment, TCR-deficient T cells described herein, isolatedpopulations thereof, or therapeutic compositions comprising the sameresult in the production of a large number of T cells that are unable torespond to allogeneic MHC antigens. Hence, these T cells may be used toreconstitute people and offer protection from infection, leading tofaster self-reconstitution of people suffering from full or partial bonemarrow ablation due to radiation exposure. In the event of acatastrophic or unexpected exposure to high doses of radiation,TCR-deficient T cells described herein having another functionalreceptor, isolated populations thereof, or therapeutic compositionscomprising the same can be infused rapidly into patients to offer somereconstitution of their immune response and growth factor production fordays to weeks until their own hematopoietic cells have reconstitutedthemselves, or until the person has been treated with an additionalsource of hematopoietic stem cells (e.g. a bone marrow transplant).

One of skill would understand how to treat cancer, infection,transplantation rejection, one or more autoimmune disorders, radiationsickness, or GVHD based on their experience with use of other types of Tcells.

In addition to the illustrative TCR-deficient chNKG2D+ T cells describedherein, it is contemplated that TCR-deficient T cells can be modified ordeveloped to express other functional receptors useful in treatment ofdiseases such as cancer or infection as described previously. Briefly,the treatment methods of the invention contemplate the use of TCR−deficient T cells expressing functional non-TCR receptors, such aschNKG2D, chimeric Fv domains, NKG2D, or any other receptor to initiatesignals to T cells, thereby creating potent, specific effector T cells.One of skill in the art can select the appropriate receptor to beexpressed by the TCR-deficient T cell based on the disease to betreated. For example, receptors that can be expressed by theTCR-deficient T cell for treatment of cancer would include any receptorthat binds to a ligand that has been identified on cancer cells. Suchreceptors include, but are not limited to, NKG2D, NKG2A, NKG2C, NKG2F,LLT1, AICL, CD26, NKRP1, NKp30, NKp44, NKp46, CD244 (2B4), DNAM-1, andNKp80.

In another embodiment of the invention, such receptors include, but notlimited to, chimeric receptors comprising a ligand binding domainobtained from NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1,NKp30, NKp44, NKp46, CD244 (2B4), DNAM-1, and NKp80, or an anti-tumorantibody such as anti-Her2neu and anti-EGFR, and a signaling domainobtained from CD3zeta, Dap10, CD28, 41BB, and CD40L.

In a further embodiment of the invention, the chimeric receptor bindsMIC-A, MIC-B, Her2neu, EGFR, mesothelin, CD38, CD20, CD19, PSA, MUC1,MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13,MUC15, MUC16, MUC17, MUC19, MUC20, estrogen receptor, progesteronereceptor, RON, or one or more members of the ULBP/RAET1 family includingULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

Also embraced by the present invention are TCR-deficient T cells thatexpress a non-TCR pathogen-associated receptor and the use of theTCR-deficient T cells expressing the pathogen receptor to treat orprevent infectious disease. In this embodiment, the non-TCR receptorbinds to virus antigen or viral-induced antigen found on the surface ofan infected cell. The infection to be prevented or treated, for examplemay be caused by a virus, bacteria, protozoa, or parasite. Viruses whichcan be treated include, but are not limited to, HCMV, EBV, hepatitistype A, hepatitis type B (HBV), hepatitis type C (HCV), ebola virus,VSV, influenza, varicella, adenovirus, herpes simplex type I (HSV-1),herpes simplex type 2 (HSV-2), rinderpest, rhinovirus, echovirus,rotavirus, respiratory syncytial virus, papilloma virus, cytomegalovirus(CMV), echinovirus, arbovirus, hantavirus, coxsackie virus, mumps virus,measles virus, rubella virus, polio virus, and/or human immunodeficiencyvirus type 1 or type 2 (HIV-1, HIV-2). Non-viral infections which can betreated using the TCR-deficient T cells include, but are not limited to,infectious Staphylococcus sp., Enterococcus sp., Bacillus anthracis,Lactobacillus sp., Listeria sp., Corynebacterium diphtheriae, Nocardiasp., Streptococcus sp., Pseudomonas sp., Gardnerella sp., Streptomycessp., Thermoactinomyces vulgaris, Treponema sp., Camplyobacter sp.,Raeruginosa sp., Legionella sp., N. gonorrhoeae, N. meningitides, F.meningosepticum, F. odoraturn, Brucella sp., B. pertussis, B.bronchiseptica, E. coli, Klebsiella, Enterobacter, S. marcescens, S.liquefaciens, Edwardsiella, P. mirabilis, P. vulgaris, Streptobacillus,R. fickettsfi, C. psittaci, C. trachornatis, M. tuberculosis, M.intracellulare, M. folluiturn, M. laprae, M. avium, M. bovis, M.africanum, M. kansasii, M. lepraernurium, trypanosomes, Chlamydia, orrickettsia.

Efficacy of the compositions of the present invention can bedemonstrated in the most appropriate in vivo model system depending onthe type of drug product being developed. The medical literatureprovides detailed disclosure on the advantages and uses of a widevariety of such models. For example, there are many different types ofcancer models that are used routinely to examine the pharmacologicalactivity of drugs against cancer such as xenograft mouse models (e.g.,Mattem, J. et al. 1988. Cancer Metastasis Rev. 7:263-284; Macor, P. etal. 2008. Curr. Pharm. Des. 14:2023-2039) or even the inhibition oftumor cell growth in vitro. In the case of GVHD, there are models inmice of both acute GVHD (e.g., He, S. et al. 2008. J. Immunol.181:7581-7592) and chronic GVHD (e.g., Xiao, Z. Y. et al. 2007. LifeSci. 81:1403-1410).

Once the compositions of the present invention have been shown to beeffective in vivo in animals, clinical studies may be designed based onthe doses shown to be safe and effective in animals. One of skill in theart can design such clinical studies using standard protocols asdescribed in textbooks such as Spilker (2000. Guide to Clinical Trials.Lippincott Williams & Wilkins: Philadelphia).

Administration

In one embodiment of the invention, the TCR-deficient T cells areadministered to a recipient subject at an amount of between about 10⁶ to10¹¹ cells. In a preferred embodiment of the invention, theTCR-deficient T cells are administered to a recipient subject at anamount of between 10⁸ to 10⁹ cells. In a preferred embodiment of theinvention, the TCR-deficient T cells are administered to a recipientsubject with a frequency of once every twenty-six weeks or less, such asonce every sixteen weeks or less, once every eight weeks or less, oronce every four weeks or less.

These values provide general guidance of the range of transduced T cellsto be utilized by the practitioner upon optimizing the method of thepresent invention for practice of the invention. The recitation hereinof such ranges by no means precludes the use of a higher or lower amountof a component, as might be warranted in a particular application. Forexample, the actual dose and schedule can vary depending on whether thecompositions are administered in combination with other pharmaceuticalcompositions, or depending on interindividual differences inpharmacokinetics, drug disposition, and metabolism. One skilled in theart readily can make any necessary adjustments in accordance with theexigencies of the particular situation.

A person of skill in the art would be able to determine an effectivedosage and frequency of administration based on teachings in the art orthrough routine experimentation, for example guided by the disclosureherein and the teachings in Goodman, L. S., Gilman, A., Brunton, L. L.,Lazo, J. S., & Parker, K. L. (2006). Goodman & Gilman's thepharmacological basis of therapeutics. New York: McGraw-Hill; Howland,R. D., Mycek, M. J., Harvey, R. A., Champe, P. C., & Mycek, M. J.(2006). Pharmacology. Lippincott's illustrated reviews. Philadelphia:Lippincott Williams & Wilkins; and Golan, D. E. (2008). Principles ofpharmacology: the pathophysiologic basis of drug therapy. Philadelphia,Pa., [etc.]: Lippincott Williams & Wilkins. The dosing schedule can bebased on well-established cell-based therapies (see, e.g., Topalian andRosenberg (1987) Acta Haematol. 78 Suppl 1:75-6; U.S. Pat. No.4,690,915) or an alternate continuous infusion strategy can be employed.

In another embodiment of the invention, the TCR-deficient T cells areadministered to a subject in a pharmaceutical formulation.

In one embodiment of the invention, the TCR-deficient T cells may beoptionally administered in combination with one or more active agents.Such active agents include analgesic, antipyretic, anti-inflammatory,antibiotic, antiviral, and anti-cytokine agents. Active agents includeagonists, antagonists, and modulators of TNF-α, IL-2, IL-4, IL-6, IL-10,IL-12, IL-13, IL-18, IFN-α, IFN-γ, BAFF, CXCL13, IP-10, VEGF, EPO, EGF,HRG, Hepatocyte Growth Factor (HGF), Hepcidin, including antibodiesreactive against any of the foregoing, and antibodies reactive againstany of their receptors. Active agents also include 2-Arylpropionicacids, Aceclofenac, Acemetacin, Acetylsalicylic acid (Aspirin),Alclofenac, Alminoprofen, Amoxiprin, Ampyrone, Arylalkanoic acids,Azapropazone, Benorylate/Benorilate, Benoxaprofen, Bromfenac, Carprofen,Celecoxib, Choline magnesium salicylate, Clofezone, COX-2 inhibitors,Dexibuprofen, Dexketoprofen, Diclofenac, Diflunisal, Droxicam,Ethenzamide, Etodolac, Etoricoxib, Faislamine, fenamic acids, Fenbufen,Fenoprofen, Flufenamic acid, Flunoxaprofen, Flurbiprofen, Ibuprofen,Ibuproxam, Indometacin, Indoprofen, Kebuzone, Ketoprofen, Ketorolac,Lomoxicam, Loxoprofen, Lumiracoxib, Magnesium salicylate, Meclofenamicacid, Mefenamic acid, Meloxicam, Metamizole, Methyl salicylate,Mofebutazone, Nabumetone, Naproxen, N-Arylanthranilic acids, NerveGrowth Factor (NGF), Oxametacin, Oxaprozin, Oxicams, Oxyphenbutazone,Parecoxib, Phenazone, Phenylbutazone, Phenylbutazone, Piroxicam,Pirprofen, profens, Proglumetacin, Pyrazolidine derivatives, Rofecoxib,Salicyl salicylate, Salicylamide, Salicylates, Sulfinpyrazone, Sulindac,Suprofen, Tenoxicam, Tiaprofenic acid, Tolfenamic acid, Tolmetin, andValdecoxib.

Antibiotics include Amikacin, Aminoglycosides, Amoxicillin, Ampicillin,Ansamycins, Arsphenamine, Azithromycin, Azlocillin, Aztreonam,Bacitracin, Carbacephem, Carbapenems, Carbenicillin, Cefaclor,Cefadroxil, Cefalexin, Cefalothin, Cefalotin, Cefamandole, Cefazolin,Cefdinir, Cefditoren, Cefepime, Cefixime, Cefoperazone, Cefotaxime,Cefoxitin, Cefpodoxime, Cefprozil, Ceftazidime, Ceftibuten, Ceftizoxime,Ceftobiprole, Ceftriaxone, Cefuroxime, Cephalosporins, Chloramphenicol,Cilastatin, Ciprofloxacin, Clarithromycin, Clindamycin, Cloxacillin,Colistin, Co-trimoxazole, Dalfopristin, Demeclocycline, Dicloxacillin,Dirithromycin, Doripenem, Doxycycline, Enoxacin, Ertapenem,Erythromycin, Ethambutol, Flucloxacillin, Fosfomycin, Furazolidone,Fusidic acid, Gatifloxacin, Geldanamycin, Gentamicin, Glycopeptides,Herbimycin, Imipenem, Isoniazid, Kanamycin, Levofloxacin, Lincomycin,Linezolid, Lomefloxacin, Loracarbef, Macrolides, Mafenide, Meropenem,Meticillin, Metronidazole, Mezlocillin, Minocycline, Monobactams,Moxifloxacin, Mupirocin, Nafcillin, Neomycin, Netilmicin,Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Oxytetracycline,Paromomycin, Penicillin, Penicillins, Piperacillin, Platensimycin,Polymyxin B, Polypeptides, Prontosil, Pyrazinamide, Quinolones,Quinupristin, Rifampicin, Rifampin, Roxithrornycin, Spectinomycin,Streptomycin, Sulfacetamide, Sulfamethizole, Sulfanilimide,Sulfasalazine, Sulfisoxazole, Sulfonamides, Teicoplanin, Telithromycin,Tetracycline, Tetracyclines, Ticarcillin, Tinidazole, Tobramycin,Trimethoprim, Trimethoprim-Sulfamethoxazole, Troleandomycin,Trovafloxacin, and Vancomycin.

Active agents also include Aldosterone, Beclometasone, Betamethasone,Corticosteroids, Cortisol, Cortisone acetate, Deoxycorticosteroneacetate, Dexamethasone, Fludrocortisone acetate, Glucocorticoids,Hydrocortisone, Methylprednisolone, Prednisolone, Prednisone, Steroids,and Triamcinolone. Any suitable combination of these active agents isalso contemplated.

A “pharmaceutical excipient” or a “pharmaceutically acceptableexcipient” is a carrier, usually a liquid, in which an activetherapeutic agent is formulated. In one embodiment of the invention, theactive therapeutic agent is a population of TCR-deficient T cells. Inone embodiment of the invention, the active therapeutic agent is apopulation of TCR-deficient T cells expressing a functional, non-TCRreceptor. The excipient generally does not provide any pharmacologicalactivity to the formulation, though it may provide chemical and/orbiological stability. Exemplary formulations can be found, for example,in Remington's Pharmaceutical Sciences, 19^(th) Ed., Grennaro, A., Ed.,1995 which is incorporated by reference.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents that arephysiologically compatible. In one embodiment, the carrier is suitablefor parenteral administration. Alternatively, the carrier can besuitable for intravenous, intraperitoneal, intramuscular, or sublingualadministration. Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions for the extemporaneous preparation ofsterile injectable solutions or dispersions. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

In a particularly preferred embodiment of the invention, appropriatecarriers include, but are not limited to, Hank's Balanced Salt Solution(HBSS), Phosphate Buffered Saline (PBS), or any freezing medium havingfor example 10% DMSO and 90% human serum.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The invention contemplatesthat the pharmaceutical composition is present in lyophilized form. Thecomposition can be formulated as a solution. The carrier can be adispersion medium containing, for example, water.

For each of the recited embodiments, the compounds can be administeredby a variety of dosage forms. Any biologically-acceptable dosage formknown to persons of ordinary skill in the art, and combinations thereof,are contemplated. Examples of such dosage forms include, withoutlimitation, liquids, solutions, suspensions, emulsions, injectables(including subcutaneous, intramuscular, intravenous, and intradermal),infusions, and combinations thereof.

The above description of various illustrated embodiments of theinvention is not intended to be exhaustive or to limit the invention tothe precise form disclosed. While specific embodiments of, and examplesfor, the invention are described herein for illustrative purposes,various equivalent modifications are possible within the scope of theinvention, as those skilled in the relevant art will recognize. Theteachings provided herein of the invention can be applied to otherpurposes, other than the examples described above.

These and other changes can be made to the invention in light of theabove detailed description. In general, in the following claims, theterms used should not be construed to limit the invention to thespecific embodiments disclosed in the specification and the claims.Accordingly, the invention is not limited by the disclosure, but insteadthe scope of the invention is to be determined entirely by the followingclaims.

The invention may be practiced in ways other than those particularlydescribed in the foregoing description and examples. Numerousmodifications and variations of the invention are possible in light ofthe above teachings and, therefore, are within the scope of the appendedclaims.

Certain teachings related to T-cell receptor deficient T-cellcompositions and methods of use thereof were disclosed in U.S.Provisional patent application No. 61/255,980, filed Oct. 29, 2009, thedisclosure of which is herein incorporated by reference in its entirety.

Certain teachings related to the production of T cells expressingchimeric receptors and methods of use thereof were disclosed in U.S.patent application publication no. US 2010/0029749, published Feb. 4,2010, the disclosure of which is herein incorporated by reference in itsentirety.

Certain polynucleotide sequences useful in the production of T-cellreceptor deficient T-cells of the invention are disclosed in thesequence listing accompanying this patent application filing, and thedisclosure of said sequence listing is herein incorporated by referencein its entirety.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books, or otherdisclosures) in the Background of the Invention, Detailed Description,and Examples is herein incorporated by reference in their entireties.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXAMPLES Example 1: Production of T Cell Receptor (TCR)-Deficient TCells

Minigenes are encoded on a retrovirus expression plasmid (e.g. pFB-neoor pSFG) containing 5′ and 3′ LTR sequences. The plasmids are packagedin a retroviral packaging cell line, such as PT67 or PG13, and viralparticles are collected once the packaging cells have grown toconfluence. T cells are then activated by PHA, anti-CD3, or anti-CD3/28mAbs for 1 to 3 days in complete medium (or serum free medium) plusrIL-2 (25 U/ml), and T cells are transduced by spinoculation at 32° C.in the presence of retronectin or polybrene. After resting for some 5 to7 hours, the cells are washed and placed in fresh medium plus IL-2 for 2to 7 days. Cells are counted periodically to avoid excessive cellconcentration (i.e., >2×10⁶ cells/ml) and re-plated at 7×10⁵ cells/ml.Selection medium to remove non-transduced T cells is optionally usedafter 2 days for a period of 3 to 5 days. Live cells are harvested byLymphoprep™ (Sentinel, Milan, Italy) gradient and further expanded for 1to 3 days.

Following incubation, cells are analyzed for expression and function ofthe TCR. Functional non-TCR receptor expression may also be analyzed atthis time, if appropriate. Flow cytometry is used to test for TCR/CD3expression using fluorochrome-labeled antibodies. Live cells are stainedwith antibodies against CD5, CD8, and CD4, in combination with anantibody against CD3ε, TCRα, TCRβ, TCRγ, or TCRδ. If the expression ofeither the CD3 or TCR genes is used, the expression of both TCR proteinsand CD3 proteins should be severely reduced compared to control vectortreated T cells. Isotype control antibodies are used to control forbackground fluorescence. To identify T cells, cells are gated on CD5,then expression of CD4, CD8, CD3, and TCR is determined. Multiplesamples are used for each treatment and appropriate compensation offluorochrome emission spectra is used. The expression of anotherreceptor (e.g. chNKG2D) is determined using specific antibodies and flowcytometry, as previously described in the art (Zhang, T. et al., (2006)Cancer Res., 66(11) 5927-5933; Barber, A. et al., (2007) Cancer Res.,67(10):5003-5008).

To test for functional deficiency of the TCR, anti-CD3 stimulation ofeffector cells is used at the end of culture to measure interferon(IFN)-gamma production after 24 hours. T cells (2×10⁵) are cultured withsoluble anti-CD3 (OKT3) mAbs in complete medium. After 24 hours,cell-free conditioned medium is collected and assayed by ELISA forIFN-gamma. Changes in TCR expression or function should be reflected inreduced IFN-gamma production.

To test for the function of the functional non-TCR, specific cytokineproduction by T cells incubated with tumor cells that do, or do not,express their specific ligand is used. For example, to test the functionof chNKG2D, 10⁵ T cells are incubated with 10⁵ P815-MICA tumor cells(ligand+), 10⁵ P815 (ligand-) cells, 10⁵ RPMI8226 cells (ligand+) or Tcells alone. After 24 hours, cell-free conditioned medium is collectedand IFN-g measured by ELISA. Chimeric NKG2D T cells produce IFN-g afterculture with ligand-expressing tumor cells (Zhang, T. et al., (2006)Cancer Res., 66(11) 5927-5933; Barber, A. et al., (2007) Cancer Res.,67(10):5003-5008). It is also possible to test cellular cytotoxicityagainst ligand+ tumor cells, as previously described in the art (Zhang,T. et al., (2006) Cancer Res., 66(11) 5927-5933). Specificity is shownusing ligand-tumor cells or specific receptor blocking mAbs.

Example 2: Production of T Cell Receptor (TCR)-Deficient T CellsExpressing chNKG2D

In this example, simultaneous expression of a chNKG2D receptor andinhibition of endogenous TCR expression is performed. In this example, amurine chNKG2D receptor is used, composed of NKG2D in combination with aN-terminally attached CD3-zeta. The chNKG2D receptor is generated andexpressed in murine T-cells. NKG2D is a type II protein, in which theN-terminus is located intracellularly (Raulet (2003) Nat. Rev. Immunol.3:781-790), whereas the CD3-zeta chain is type I protein with theC-terminus in the cytoplasm (Weissman, et al. (1988) Proc. Natl. Acad.Sci. USA 85:9709-9713). To generate a chimeric NKG2D-CD3-zeta fusionprotein, an initiation codon ATG is placed ahead of the coding sequencefor the cytoplasmic region of the CD3-zeta chain (without a stop codonTAA) followed by a wild-type NKG2D gene. Upon expression, theorientation of the CD3-zeta portion is reversed inside the cells. Theextracellular and transmembrane domains are derived from NKG2D. A secondchimeric gene encoding the Dap10 gene followed by a fragment coding forthe CD3-zeta cytoplasmic domain is also constructed. FIG. 1 presents thestructures of the chimeric and wild-type receptors.

An shRNA is operably linked in a lentiviral vector with the chNKG2Dreceptor. To achieve expression of both genes, the shRNA is driven by aU6 promoter and the chNKG2D receptor by a PGK promoter. Primary humanPBMCs are isolated from healthy donors and activated with low-dosesoluble anti-CD3 and 25 U/ml rhulL-2 for 48 hours. Although it is notrequired to activate T cells for lentiviral transduction, thetransduction will work more efficiently and allow the cells to continueto expand in IL-2. The activated cells are washed and transduced using a1 h spin-fection at 30° C., followed by a resting period for 7 h. Thecells are washed and cultured in 25 U/ml IL-2 for 3 to 7 d to allowexpansion of the effector cells in a similar manner as we do for use ofthe cells in vivo. The expression of TCRα3, CD3, and NKG2D is evaluatedby flow cytometry and quantitative realtime-PCR (QRT-PCR). The number ofCD4+ and CD8+ T cells are determined by flow cytometry. Overall cellnumbers and the percentage of TCR complex deficient and expressing Tcells are determined by flow cytometry. These are compared to PBMCs thatare transduced with the shRNA or chNKG2D genes alone (as controls).Vector-only transduced cells are also included as controls.

It is anticipated that those cells with no or little TCR expression atthe cell surface will express higher amounts of cell surface NKG2Dbecause of co-expression of the chNKG2D receptor.

As an alternative, transduction may occur with two viruses at the sametime, one with the shRNA construct and one with the chNKG2D receptor. Alarger amount of the chNKG2D virus is used to ensure high expression ofchNKG2D in those T cells that lack TCR expression. TCR+ T cells that mayremain are removed to obtain TCR−, chNKG2D+ T cells.

After viral transduction and expansion, the TCR+ and TCR− cells areseparated by mAbs with magnetic beads over Miltenyi columns.Verification of chNKG2D expression is performed by QRT-PCR usingspecific primers for the chNKG2D receptor.

To determine whether the T cells have lost TCR function and retainedchNKG2D function, transduced or control T cells are cultured withmitomycin C-treated allogeneic PBMCs or syngeneic PBMCs. The T cells arepreloaded with CFSE, which is a cell permeable dye that divides equallybetween daughter cells after division. The extent of cell division canbe easily determined by flow cytometry.

To determine whether the shRNA construct can inhibit TCR function andallow chNKG2D receptor function, transduced T cells are cultured withmitomycin C-treated allogeneic PBMCs, syngeneic PBMCs, or tumor cells:P815-MICA (a murine tumor expressing human MICA, a ligand for NKG2D),P815, A2008 (a human ovarian tumor cell, NKG2D ligand+), and U266 (ahuman mycloma cell line, NKG2D ligand+). After 48 hours, cell-freesupernatants are collected and the amount of IL-2 and IFN-γ producedwill be quantitated by ELISA. T cells alone are used as a negativecontrol.

Example 3: In Vivo Administration of T Cell Receptor (TCR)-Deficient TCells Expressing chNKG2D

In this example, the TCR-deficient T cells expressing a murine chNKG2Dreceptor as produced in Example 2 are administered to mice to evaluatethe in vivo therapeutic potential of said T cells on certain cancers.The chimeric NKG2D-bearing T cells (106) are co-injected with RMA/Rae-1βtumor cells (10⁵) subcutaneously to C57BL/6 mice. ChimericNKG2D-bearing, TCR-deficient T cell-treated mice that are tumor-free orhave tumor-inhibited growth of RMA/Rae-1β tumors after 30 days reflectstherapeutic anti-cancer activity in these mice.

In a second and more stringent model, transduced T cells (10⁷) areadoptively transferred i.v. into B6 mice one day before RMA/Rae-1β s.c.tumor inoculation in the right flank. Suppression of the growth of theRMA/Rae-1β tumors (s.c.) compared with control vector-modified T cellsreflects therapeutic anti-cancer activity in these mice. As for toxicityof treatment with chimeric NKG2D-modified T cells, it is anticipatedthat the animals will not show any overt evidence of inflammatory damage(i.e., ruffled hair, hunchback or diarrhea, etc.) when treated withchimeric NKG2D-bearing T cells, which would be reflective of a lack ofovert toxicity.

In a more stringent model of established ovarian tumors (ID8),transduced chNKG2D T cells (5×10⁶ T cells, i.p.) are injected into micebearing tumors for 5 weeks. Mice are further injected with T cells at 7and 9 weeks following tumor challenge. Under these conditions, micetreated with chNKG2D T cells will remain tumor-free for more than 250days, whereas mice treated on a similar schedule with control T cellswill die from tumor growth within 100 days. As for toxicity of treatmentwith chimeric NKG2D-modified T cells, it is anticipated that the animalswill not show any overt evidence of inflammatory damage (i.e., ruffledhair, hunchback or diarrhea, etc.) when treated with chimericNKG2D-bearing T cells, which would be reflective of a lack of overttoxicity.

In a model of multiple myeloma, mice bearing 5T33MM tumor cells aretreated on day 12 post tumor cell infusion with chNKG2D T cells (5×10⁶cells, i.v.). This treatment will result in an increased life-span ofall mice and about half of these mice will be long-term, tumor-freesurvivors. Mice treated with control T cells will succumb to theirtumors within 30 days. No overt evidence of toxicity will be observeddue to treatment with the chNKG2D T cells.

Because the immune system can select for tumor variants, the mosteffective immunotherapies for cancer are likely going to be those thatinduce immunity against multiple tumor antigens. In a third experiment,it is tested whether treatment with chimeric NKG2D-bearing T cells willinduce host immunity against wild-type tumor cells. Mice that aretreated with chimeric NKG2D-bearing T cells and 5T33MM tumor cells, andare tumor-free after 80 days, are challenged with 5T33MM tumor cells.Tumor-free surviving mice are resistant to a subsequent challenge of5T33MM cells (3×10⁵), compared to control naive mice which succumb tothe tumor within an average of 27 days. However, tumor-free survivingmice are not resistant to a subsequent challenge of RMA-Rae1 tumor cells(3×10⁵), and succumb to the tumor in a similar time-span as naïve mice(20 days). This indicates that adoptive transfer of chimericNKG2D-bearing T cells will allow hosts to generate tumor-specific T cellmemory.

1-35. (canceled)
 36. A method of producing one or more compositionscomprising human T cells which express a heterologous ligand bindingdomain or a heterologous receptor other than a T cell receptor (TCR),wherein said human T cells are derived from primary human T cells andwherein said method comprises: (i) obtaining one or more compositionscomprising isolated primary human T cells, each of said compositionsbeing comprised of primary human T cells isolated from at least onehuman donor; (ii) modifying the primary human T cells comprised in saidone or more compositions comprising primary human T cells in order tofunctionally impair or reduce expression of one or more components ofthe TCR complex endogenously expressed by said human primary T cells;(iii) further modifying the primary human T cells in the one or morecompositions comprising human T cells by introducing a nucleic acidwhich encodes a desired ligand binding domain or a desired receptor,wherein said ligand binding domain and receptor do not comprise a TCR;thereby producing one or more compositions comprising human T cellswhich express at least one desired ligand binding domain or desiredreceptor and further wherein one or more components of the TCR complexendogenously expressed by said human primary T cells is functionallyimpaired or the expression of one or more components of the TCR complexendogenously expressed by said human primary T cells is reduced.
 37. Themethod of claim 36, wherein said desired ligand binding domain ordesired receptor binds to tumor cells.
 38. The method of claim 36,wherein said desired ligand binding domain or desired receptor binds toinfected cells.
 39. The method of claim 36, wherein said desired ligandbinding domain or desired receptor binds to autoimmune cells.
 40. Themethod of claim 36, wherein said T cells are further modified by theintroduction of a nucleic acid encoding a signaling domain.
 41. Themethod of claim 36, wherein reduced expression of the targeted TCRcomponent or the functional impairment or reduced expression of theendogenous TCR by the T cells in said one or more compositions obtainedafter effecting steps (i)-(iii) is confirmed by effecting one or more ofthe following procedures: (a) detecting the level of expression of saidtargeted TCR component in T cells comprised in said one or morecompositions, (b) detecting the proliferation of T cells obtained insaid one or more compositions in the presence of HLA-mismatched PMBCsand (c) detecting the level of gamma interferon produced by T cellscomprised in said one or more compositions in response to HLA-mismatcheddonor cells.
 42. The method of claim 36, wherein said one or morecompositions comprising human T cells are derived from different humandonors.
 43. The method of claim 36, wherein said one or morecompositions comprising human T cells are derived from the same humandonor.
 44. The method of claim 36, wherein said one or more componentsof the TCR complex include TCR-α, TCR-β, TCR-γ, TCR-δ, CD3-γ, CD3-δ,CD3-ζ, and CD3-ε.
 45. The method of claim 36, wherein in step (i) thefunctional impairment or reduced expression of the one or morecomponents of the TCR complex is effected by introducing into theprimary human T cells at least one short hairpin RNA (shRNA) whichtargets a nucleic acid that encodes at least one component of afunctional TCR expressed by said primary human T cells.
 46. The methodof claim 45, wherein said one or more components of the TCR complexinclude TCR-α, TCR-β, TCR-γ, TCR-δ, CD3-γ, CD3δ, CD3-ζ, and CD3-ε. 47.The method of claim 36, wherein the compositions are in a form wherebythey may be stored and can be used for future therapy.
 48. The method ofclaim 47, wherein the compositions are in a form whereby they may bestored and can be used for future therapy.
 49. The method of claim 36,wherein the primary human T cells express CD4 or CD8.
 50. The method ofclaim 49, wherein the primary human T cells express CD4 or CD8.
 51. Themethod of claim 36, wherein the primary human T cells of (i) comprisehuman peripheral blood mononuclear cells or cytotoxic T cells.
 52. Themethod of claim 51, wherein the primary human T cells of (i) comprisehuman peripheral blood mononuclear cells or cytotoxic T cells.
 53. Themethod of claim 36, wherein the ligand binding domain is obtained fromNKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26 or NKRP1.
 54. The method ofclaim 36, wherein the ligand binding domain is obtained from ananti-tumor chimeric antigen receptor or anti-tumor antibody.
 55. Themethod of claim 40, wherein the signaling domain is obtained from CD3 ζ.56. The method of claim 36, wherein the receptor binds to a ligandexpressed on tumor or infected cells.
 57. The method of claim 36,wherein the receptor binds to any one of MIC-A, MIC-B, an estrogenreceptor, a progesterone receptor, RON or one or more members of theULBP family.
 58. The method of claim 57, wherein the one or more membersof the ULBP family include Rae-1, H-60, HCMV UL18, Rae-1β, ULBP2, andULBP3.
 59. The method of claim 36, further comprising engineering theprimary human T cells to express another receptor which initiates asignal to T cells.
 60. The method of claim 36, wherein the receptorcomprises a ligand binding domain from NKG2D.
 61. One or morecompositions comprising human T cells produced according to claim 36.62. A method of treatment comprising the administration of one or morecompositions comprising human T cells produced according to claim 36.63. The method of claim 62 which is used to treat a subject with canceror an infectious condition.